Human hepatoma lines, methods for obtaining same and uses thereof

ABSTRACT

Human hepatoma cell lines are provided that comprises a set of cells clonally derived from a cancerous human liver cell, in which each cell of the set expresses a receptor having an affinity for binding a virus of the  Flaviviridae  genus and a virus of the  Hepadnaviridae  genus to enable infection by viruses of both genus in native forms, and in which each cell of the set exhibits susceptibility to infection by a hepatotropic parasite of the  Leishmania  genus in a native form. Various compositions and methods relating to diagnostic, therapeutic, and prophylactic embodiments are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national phase of PCT/FR02/002391 filed Jul. 8,2002, which claims priority of the French Application No. 01/09044,filed on Jul. 6, 2001, which are hereby incorporated by reference intheir entireties and are relied upon.

The invention relates to novel human hepatoma lines. It also relates tomethods for obtaining same and their uses in diagnostic, therapeutic andprophylactic applications.

INCORPORATION-BY-REFERENCE & TEXTS

The material on the accompanying compact disc is hereby incorporated byreference into this application. The accompanying compact disc containsone file, ALX1 GENERAL-#1308566-v1-03247515 TXT.TXT created on Feb. 13,2008, is 1.2 KB. The file can be accessed using Microsoft Word on acomputer that uses Windows OS.

Hepatitis B is an infectious disease which is widespread throughout theworld. Its virus (abbreviated to HBV) is a small virus with DNApossessing a high host specificity. In fact, only humans and the higherprimates are infected by this virus, which strongly limits the in vivostudy models of this infection. However, a “duck” model exists whichallows the study of an entire replication cycle of a hepadnavirus (invitro and in vivo). This hepadnavirus, although close to the HBV virus,however has a somewhat different biology. Moreover, the metabolicbehaviour of the duck cannot be simply compared to that of humans.

HBV moreover shows a strong tropism for the liver and preferentiallytargets the parenchymatous cells. Also, unlike other viruses, only thedifferentiated hepatic cells can be infected.

A cell line derived from hepatomas has allowed rapid progress in theunderstanding of the replication mechanisms. This line, which is widelyused, is referenced under the name HepG2. A derived clone HepG2 2.2.15has the advantage of possessing a considerable ability to proliferateand actively replicate the virus. However, this replication is possibleonly after the introduction of the viral DNA into the cells, bytransfection. Natural infection of this line by the HBV has provedimpossible. Moreover, due to the age of this line, the karyotype of itscells is broadly modified, which makes it an imperfect model formimicking human hepatocytes. Finally, the differentiation capacity ofthese cells remains fairly limited.

Other more or less differentiated cell lines have been proposed. Inparticular the line Hep3B, which is slightly different from the lineHepG2, and which has the same limits as the latter can be mentioned. Allthe line infection tests have proved negative or disappointing in termsof effectiveness.

In order to study the mechanisms of HBV infection, a model of humanhepatocytes in primary culture has been developed. This model is veryuseful in involving human hepatocytes and allowing access to the wholeviral cycle. However, it is difficult and tedious to manipulate, andobtaining bioptic fragments is increasingly difficult and random.

The inventors' work in this field has led them to record that humanhepatoma lines, actively multiplying whilst being capable of beinginfected by parasites and/or viruses, in particular HBV, could beobtained by carrying out a cell selection under specific conditions.

The invention therefore aims to provide new human hepatoma lines capableof being infected by parasites and/or viruses, and the cells or elementsof these cells originating from these lines.

It also relates to a process for selecting such lines.

A subject of the invention is also the uses of these lines indiagnostic, therapeutic and prophylactic applications.

According to the present invention, the human hepatoma cell lines arecharacterized in that they are capable of being infected naturally byparasites and/or viruses; said parasites may or may not be hepatotropic,such as Plasmodium or parasites of the genus leishmania; and expressreceptors of the family of the Flaviviridae and Hepadnaviridae viruses,preferably HBV and HCV.

These new lines thus allow complete study of the cycle of the parasitesand/or viruses in particular the viral cycle of the HBV, from thenatural infection stage to the replication and/or propagation of thevirus. It will be observed in this respect that no human hepatoma cellline is at present capable of being infected by Plasmodium falciparum,i.e. that no human hepatoma cell line is capable of supporting thedevelopment of mature forms of this hepatotropic parasite (schizonts).Moreover, due to its ability to proliferate, these lines are very easyto manipulate.

According to another aspect of the invention, these lines are capable ofreaching an advanced level of differentiation.

In particular, the cells resulting from the lines according to theinvention make it possible to reach a stage of hepatic differentiation,i.e. a morphology close to cells constituting the liver, such as thehepatocytes and/or the biliary cells with in particular:

-   -   the formation of trabeculae of parenchymatous cells,    -   the formation of functional biliary canaliculi, with at cell        level, the reconstitution of a biliary pole.

Throughout the Application, reference is made to typical notions of thestructural organization of the liver cells. These notions, in particularthe biliary pole, are explained in the preamble to the “Results”paragraph of Example 1 hereafter. It is surprising to note that thecells originating from the lines according to the invention are capableof practically identically mimicking the structural organization of theliver cells and of reproducing its functions: in fact, the biliarycanaliculi formed by the cells of the lines according to the inventionare functional, i.e. capable of playing their detoxification role. Thisfunctional differentiation is moreover highly advanced: the cells of thehuman hepatoma cell lines according to the invention can express thefunctions characteristic of the hepatocyte, namely:

-   -   the production of plasmatic proteins, in particular albumin, and        transferrin,    -   the detoxification function, in particular:        -   the expression of various forms of P450 cytochromes, such as            CYP2E1, CYP3A and/or CYP1A, the expression of various forms            of detoxification phase II enzymes in particular GSTα,        -   the conjugation of biliary salts,        -   the elimination of urea.    -   the energy regulation function:        -   the storage of sugar in the form of glycogen and the            production of glucose by glycolysis, in particular, the            expression of aldolase B, and/or neoglucogenesis,        -   metabolism of lipids.

It will be noted with interest that no human hepatoma cell line wascapable of producing cells which could reach a level of differentiationsuch that these cells could express practically all of the functions ofthe normal human hepatocyte, in particular, the detoxification functionsof phases I and II (CYP2E1, CYP3A, CYP1A and GSTa).

According to an unexpected aspect, the cells originating from the linesaccording to the invention possess properties of pluripotent cells, inparticular properties of resident and/or oval stem cells, i.e. capableof differentiating towards the hepatocyte, biliary, pancreatic and/orintestinal route.

The resident stem cells are liver cells, capable, during a massivedestruction of the liver, of actively multiplying in order to regeneratethe part destroyed. These cells can evolve towards a hepatocyte orbiliary lineage (FIG. 1). Moreover, these resident stem cells, sometimesalso designated oval cells, have the ability to differentiate intodifferent cell types such as pancreatic, intestinal and/or hepaticcells.

Another advantage of the lines according to the invention lies in theirability to proliferate actively. Thus, in the proliferation phase, thecell population doubles in approximately 24 hours.

The invention relates in particular to the cell line filed on 5 th Apr.2001 at the Collection Nationale de Cultures de Microorganismes,Institut Pasteur, 25 rue du Docteur Roux, F-75724 Paris Cedex 15, underNo. I-2652.

This line, called HepaRG, is an example of a cell line having all thecharacteristics of the lines according to the invention at the sametime: it can be infected naturally by parasites, viruses, it has themorphological characteristics set forth above, and possesses all thebiological functions of a hepatic cell. It appears to be a virtuallyperfect model of the hepatic cells.

Also included within the scope of the invention are cells or elements ofcells originating from the lines according to the invention and inparticular, membranes, receptors and/or antigens originating from thismembrane, cytoplasm, nucleus, genes and/or gene products, DNA, mRNA,cDNA, proteins, peptides.

Moreover, the invention aims to provide methods for obtaining andselecting such lines.

This involves in particular a process for selecting human hepatoma celllines comprising:

-   -   a phase of cell proliferation in a culture medium comprising        continuously at least one cortico-steroid at a non-toxic        concentration which promotes the differentiation of normal human        hepatocytes, and in particular their optimum differentiation.    -   then, having reached confluence, a phase of cell differentiation        in this same medium, by the addition of DMSO, in a quantity        sufficient to induce differentiation,    -   this process being repeated several times, preferentially 3        times, if desired.

This cell selection process is indispensable for maintaining theproperties of the lines according to the invention. In fact, thisselection process induces high cell mortality. Example 3 belowdemonstrates the necessary cooperation between the cortico-steroid andthe DMSO.

The phrase “non-toxic concentration which promotes the differentiationof normal human hepatocytes”, is used to refer to the cortico-steroidconcentration promoting, during its addition to a culture of normalhuman hepatocytes, the differentiation of the cells towards a morphologyand a functional state described in the preamble to the “Results”paragraph of Example 1. This concentration is non-toxic, i.e. itsaddition does not lead to a cell mortality rate greater thanapproximately 10%.

Moreover, the term “quantity sufficient to induce the differentiation”is used to refer to the quantity of DMSO necessary to induce thedifferentiation of a culture of normal human hepatocytes.

It will be noted with interest that a preferential process comprises acortico-steroid present at a high concentration, continuously in themedium, in contrast to the processes usually used in hepatoma culture.Surprisingly, the presence of this cortico-steroid in no way preventsthe lines from proliferating.

This selection process has made it possible to develop a process forobtaining the lines according to the invention comprising a stage ofbiopsy of a solid tumor of hepatocarcinoma type, a stage of isolation ofthe cell population using a proteolytic enzyme and a stage of selectionof the lines using the selection process detailed above.

The proteolytic enzyme preferentially used is trypsin and/orcollagenase.

The invention also proposes a process for infecting hepatic cells with ahepatotropic parasite and/or a virus comprising:

-   -   a selection phase using the abovementioned selection process,    -   a differentiation phase allowing the cells to reach a morphology        close to the hepatocyte using culture medium comprising at least        one cortico-steroid at a non-toxic concentration which promotes        optimum differentiation of normal human hepatocytes, with DMSO        added to it, in a quantity sufficient to induce differentiation,    -   an infection phase with incubation of the hepatic cells in a        culture medium comprising at least one cortico-steroid at a        non-toxic concentration which promotes optimum differentiation        of normal human hepatocytes, to which the infectious source is        added.

The hepatotropic parasite can be a Plasmodium and the virus, HBV or HCV.

A possible infectious source is the supernatant of HepG2 cells and/or apatient's serum.

The culture media used for the selection, obtaining and infectionprocesses preferably contain insulin in a quantity sufficient to promotethe survival of normal human hepatocytes, preferentially from 2.5 μg/mlto 10 μg/ml, in particular of the order of 5 μg/ml.

The insulin makes it possible to considerably improve the viability ofthe cells.

Moreover, the cortico-steroids of these culture media are preferablyhydrocortisone hemisuccinate and dexamethasone. Other inducers ofdifferentiation can be used, in particular retinoic acid and/or itssynthetic analogues, oestrogens and thyroid hormones.

The term “synthetic analogues” is used to refer to the analogues of saidcortico-steroids and retinoids of non-natural origin.

The corticoid concentration is from 10⁻⁷ M to 10⁻⁴ M, preferentiallyapproximately 5.10⁻⁵ M for the hydrocortisone hemisuccinate andapproximately 10⁻⁵ M for the dexamethasone. Finally, the DMSOconcentration, when the latter is added to the culture media, is from 1%to 4%, preferentially approximately 2%.

The invention also relates to a process for transfection of the linesaccording to the invention using a vector comprising the completesequence and/or part of genetic material from the HBV and/or HCVviruses. It also relates to a high-flow-rate screening process ofdifferentially expressed genes using a chip-type tool produced from thelines and/or cells and/or parts of cells according to the invention,preferably, these genes being differentially expressed under the effectof culture conditions, exposure to a molecule and/or virus and/or aparasite.

An example of a chip-type tool is given in Example 7 hereafter. It is acDNA chip the construction of which allows high-flow-rate screening ofdifferentially expressed genes.

The invention finally covers the use of the different culture mediaallowing the obtaining, maintaining, proliferation, selection,differentiation, transfection, infection of the lines according to theinvention. It involves in particular the use of a culture mediumcomprising continuously at least one cortico-steroid, preferentiallyhydrocortisone hemisuccinate at a non-toxic concentration which promotesoptimum differentiation of normal human hepatocytes, in order tomaintain the stability of the cell population lines and/or of the cellsaccording to the invention; as well as the use of a culture mediumcontinuously comprising at least one cortico-steroid, preferentiallyhydrocortisone hemisuccinate at a non-toxic concentration which promotesoptimum differentiation of normal human hepatocytes, with DMSO added ina quantity sufficient to induce differentiation of the cells originatingfrom the lines according to the invention; and use of the culture mediumcontinuously comprising at least one cortico-steroid at theabove-mentioned concentration as well as sodium butyrate at aconcentration sufficient to induce a biliary-type differentiation,preferably a concentration of 2.5 to 5 mM, in particular approximately3.75 nM.

As already indicated, the lines according to the invention are capableof evolving towards distinct differentiation routes. The differentiationroute is strongly influenced by the choice of culture medium. Evolutiontowards the hepatic, biliary and pancreatic routes are illustrated byExample 4.

Human hepatoma lines according to the invention are the subject of anumerous applications, given their high differentiation capacity, theirconsiderable functionality and their ease of manipulation.

A first useful application corresponds to the use of the lines and/orcells originating from the lines according to the invention, formetabolic and/or toxicity tests intended for the evaluation of newmedicaments and/or nutritional constituents and/or environmentalpollutants.

In fact, the lines according to the invention are at present the bestmodel mimicking normal human hepatocytes, in particular in terms ofdetoxification.

In particular the lines according to the invention allow the manufactureof an extracorporeal bioreactor for the transient treatment of acutehepatocellular insufficiencies. The invention also covers the use of thelines according to the invention for the screening and/or manufacture ofnew vaccines and/or antiviral molecules, in particular for the screeningof molecules active vis-à-vis one of the viral cycle stages; for themanufacture of antibodies directed against a virus belonging to theFlaviviridae and Hepadnaviridae family, in particular directed againstthe HBV and HCV and/or their cell membrane receptors; for carrying outviral neutralization and/or vaccinal composition tests comprising atleast viral particles and/or polypeptides obtained after infectionand/or transfection of the lines according to the invention, combinedwith a pharmaceutically acceptable vehicle and/or excipient and/oradjuvant.

The lines according to the invention are advantageously used for thepurposes of validating the virucidal capacity of disinfectant chemicalproducts.

The invention also proposes a new method for evaluating the virucidalcapacity of a disinfectant chemical product for cleaning equipment,premises and/or surfaces comprising:

-   -   bringing the viruses into contact with said equipment, premises        and/or surfaces,    -   the disinfecting of said equipment, premises and/or surfaces        with said disinfectant chemical product,    -   then the contamination of cells according to the invention, by        the viruses having survived the disinfecting.

Other characteristics and advantages of the invention are given in theexamples which follow. They relate, for illustration purposes, to theline I-2652 deposited at the CNCM, called HepaRG.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1, a diagrammatic representation of the different cell typesparticipating in the homeostasis of the liver,

FIG. 2, a diagrammatic cross-section of the liver, clearly showing thestructural organization of the different cells constituting the liver,

FIG. 3, the growth curve of the HepaRG line,

FIG. 4, phase contrast microphotographs of HepaRG cells at differentstages of differentiation.

FIG. 5, electron micrographs of HepaRG cells

FIG. 6, the karyotype, with RHG-type staining, of a pseudodiploidmetaphase representative of the HepaRG line,

FIG. 7, a Northern blot analysis of the expression of the mRNAs in 2liver biopsies, HepG2 cells and HepaRG cells,

FIG. 8, the effects of activity inducers CYP1A (phenacetin deethylase)and CYP3A4 (nifedipine oxidase),

FIG. 9, the influence of corticoids and DMSO on the level of hepaticmRNAs in the HepaRG cells,

FIG. 10, the effects of different factors influencing the infectabilityof the cells, and

FIG. 11, the results of infection of the HepaRG cells with HBV.

FIGS. 12 and 13, biliary and pancreatic differentiations,

FIGS. 14 and 15, the infectability of HepaRG cells with the serum ofpatients carrying HCV, in the presence or absence of interferon α,

FIG. 16, the kinetics of replication or inhibition of HCV, by interferonα,

FIG. 17, the kinetics of infection by parasites of the genus Leishmania,and,

FIGS. 18 and 19, optimizations of the protocol of infection by parasitesof the genus Leishmania.

EXAMPLE 1 Isolation of Hepatoma Cell Line

1. Materials and Methods

Cells were isolated from a liver tumor taken from a patient sufferingfrom a hepatocarcinoma and viral hepatitis C. The whole procedure wascarried out in accordance with French law and regulations and approvedby the Comite National d'Ethique, and confidentially.

The samples were cut into fine sheets, then rinsed with a HEPES-basedbuffer solution [(pH 7.7; 140 mM NaCl, 2.68 mM KCl, 0.2 mM Na₃HPO₄ and10 mM HEPES], and digested with 0.025% of collagenase D (BoehringerMannheim) diluted in the same buffer solution with 0.075% of CaCl₂ added(addition under gentle stirring at 37° C.). After two washings with theHEPES buffer solution, the cells are resuspended in a Williams' mediumE, supplemented with 10% of foetal calf serum (FCS), 100 U/ml ofpenicillin, 100 μg/ml of streptomycin, 5 μg/ml of insulin, 2 mM/ml ofL-glutamine and 5.10⁻⁷ M of hydrocortisone hemisuccinate. The cellsuspension is then distributed into different wells on a plasticsupport. After several weeks, the cell growth is sufficient to produce aculture. Its population is heterogenous, but the cells are highlydifferentiated and have a hepatocyte-type morphology. The wells havingthe most homogenous cell populations are separated with trypsin, thenredistributed. After 3 passages, the cells are aliquoted, then frozen inthe culture medium with 10% of DMSO added, and preserved in liquidnitrogen. After thawing, the well having the greatest proportion ofcells having a hepatocyte-type morphology is selected. The cells arecultured in the culture media used for their isolation and/or in thedifferentiation medium used to complete the cell selection.

In order to obtain a more frequent hepatocyte differentiation in theline, the HepaRG cells originating from the first selection are culturedin a Williams' medium E, with 5 μg/ml of insulin, 100 U/ml ofpenicillin, 100 μg/ml of streptomycin, 5.10⁻⁷ M of hydrocortisonehemisuccinate, 2 mM/ml of L-glutamine and 10% of FCS added.

The cells are subjected to a passage every 10-15 days, at a ⅕ dilution.The differentiation phase takes place in two stages:

-   -   the cells are maintained in their growth medium for two weeks,        confluence being reached at the end of one week,    -   they are then maintained in a differentiation medium        (corresponding to the preceding culture medium, with 2% of DMSO        added) for another two weeks, the medium being replenished every        2 or 3 days.        Cytogenetic Analysis

The karyotype of the HepaRG cells was analyzed after 8 passages. Thecells were first maintained for 24 to 48 hours in RPMI 1640 with 10% ofFCS added, then blocked in metaphasis by exposure to Colcemide (10μg/mL) for 45 minutes.

The cells were then treated with a hypotonic solution (0.1 M of MgCl₂),fixed with Carnoy's acetic solution and the chromosomes revealed by RHGstaining.

2. Results

Preamble

FIG. 2 shows a diagrammatic cross-section of the liver clearly showingthe structural organization of the liver cells. In particular, a normalhuman hepatocyte has the following morphological characteristics:

-   -   These are cells with a granular cytoplasm due to the fact that        they are rich in organites such as mitochondria and rough        endoplasmic reticulum vesicles, and a round and regular central        nucleus with a very dense nucleolus.    -   These cells become grouped and organized into typical trabecula,        most often formed from cords of 2 to 4 cells, interlinked by        joining structures of desmosome type, and communicating        structures of “Gap”-type, and bordered on either side by        sinusoids in which the blood circulates. This organization into        trabeculae determines the double polarity which characterizes        the hepatocyte: a sinusoidal pole on the side of the sinusoids        and a biliary pole at the interface of the hepatocytes.

The biliary pole is associated with the detoxification function and moreparticularly with the elimination of biliary salts. It is a dilatedintercellular space, closed by characteristic complex junctions (tightjunctions plus desmosomes) which delimit for each cell a specializedmembrane zone on the one hand, at the functional level by the expressionof specific proteins, and on the other hand, at the morphological levelby the formation of numerous villi.

Moreover, the term biliary cells refers to cells constituting thebiliary canals and canaliculi. The latter make it possible to evacuateinto the bile, the biliary salts originating from the hepatocytes andhaving circulated along the trabecula of parenchymatous cells via thebiliary pole.

Selection of Cells Having a Hepatocyte Morphology

The cells originating from the wells initially selected for their highproportion of hepatocyte-type cells are maintained in a culture mediumcontaining 5.10⁻⁷ M of hydrocortisone hemisuccinate. It is noted thattheir morphology becomes more and more heterogenous and removed fromthat of a hepatocyte.

In order to find a hepatocyte-type cell population, in accordance withthe invention the following selection method is then used:

-   -   a differentiation medium is used, formed from a basic medium        containing only 5 mg/L of insulin and 10% of FCS to which 2% of        DMSO and 5.10-5 M of hydrocortisone hemisuccinate are added.        Once confluence is well established, the HepaRG cells are        cultured in this medium.

In the following 2 to 4 days, the medium, which is highly toxic, causesthe death of more than 90% of the cells. In parallel, the morphology ofcertain surviving cells changes considerably.

After two weeks of this treatment, the mortality rate of the cellsappears to be zero, and certain surviving cells have the same morphologyas normal human hepatocytes, cultured under the same conditions.

It is therefore established that the differentiated cells are moreresistant to DMSO than the others.

The cells, once replaced in the medium without DMSO, return to an activeproliferation phase.

The selection process is continued. After 3 selection stages in thisdifferentiation medium, most of the cells become resistant to DMSO andcapable of re-differentiating, once confluence is reached.

The homogeneity of the population is further increased by means of twoother selection protocols.

We observed and took advantage of the fact that treatment with trypsintends to separate the most differentiated cells in the form ofmulti-cell clusters whilst the less differentiated cells are isolated.The purification of the large aggregates makes it possible to enrich thepopulation with cells capable of differentiating.

The use of collagenase is based on a very different principle: thetreatment carried out with the collagenase involves selective separationof the most differentiated cells which can be recovered and reseeded.

Finally, the stability of the phenotype of the line is considerablypromoted by the continuous use of a high concentration of hydrocortisonehemisuccinate (5.10-5 M).

Morphological Modifications: from Proliferation to Differentiation

An unexpected characteristic of the cells selected concerns theirability to proliferate in the presence of strong corticoidconcentrations. After separation, most of the cells attach to thesupport in two hours and begin to proliferate. During the exponentialproliferation phase, the population doubles in 24 hours then, when thecells reach confluence, growth slows down. The latter reaches a plateauin the 10 days following the passage, after which the cells can nolonger divide, but stay alive for several weeks.

In FIG. 3, the cells cultivated in the absence—▪— or in the presence ofhydrocortisone (5.10⁻⁵ M)—♦— for 10 passages were seeded at a density of40,000 cells per 4 cm² well and maintained in the same culture medium.The cells were collected by trypsinization and counted manually.

During the proliferation phase, the HepaRG cells form a homogenouspopulation of epithelial phenotype (absence of regular structuralorganization) and shapes different from that of the hepatocytes (FIG.4A, HepaRG cells under proliferation conditions). Once confluence isreached, the cells undergo considerable morphological modifications. Infour weeks, a number of colonies of granular hepatocyte-type cellsappear, whilst at the periphery, epithelial cells can be distinctly seen(FIG. 4B, HepaRG cells maintained at confluence for 20 days). Theaddition of DMSO two weeks after the passage induces a very completemorphological differentiation: organization of the cells into trabeculaecomparable to those obtained in primary culture of normal hepatocytes,in which canaliculus-type structures can be recognized (indicated by ablack arrow in FIG. F4). FIGS. 4C and 4D show HepaRG cells maintained atconfluence for 5 days, then treated with 2% of DMSO for 15 days(scale=100 μm).

Some epithelial cells occupy the free spaces (flat, regular cells,written in full in FIG. 4C and annotated by the letter E in FIG. 4D).The characteristic hepatocyte morphology is also translated at the levelof the organization into trabeculae of the hepatocyte cords. It isobtained at the end of two weeks of exposure to DMSO, the granular cellsthen very strongly resembling hepatocytes (FIG. 4C, cells annotated bythe letter H in FIG. 4D). At the end of these two weeks, no furthersignificant modifications are noted.

The conditions making it possible to obtain complete hepatocytedifferentiation are therefore defined by:

-   -   one week of confluence in the presence of corticoid (5.10⁵ M of        hydrocortisone),    -   then two weeks of differentiation in the presence of        hydrocortisone and 2% of DMSO.

Surprisingly, it was observed that by selectively collecting thegranular cell population from the trabeculae, the latter was capable,even after a long period of quiescence, of returning to the activeproliferation phase and once again giving rise to two hepatocyte andepithelial cell types, when confluence is reached.

An electron microscope study revealed the structural organization of thecells: two adjacent cells are strongly attached to each other bydesmosomes (FIG. 5A, view with low magnification), with structuresstrongly resembling biliary caniculi, and surrounded by complexestypical of junctions (“tight” junctions+desmosomes). FIGS. 5B and 5Cshow views with a greater magnification, showing a typical accumulationof collagen granules and a biliary canaliculus-type structure. (Scale=2μm).

In FIG. 5A, characteristic regular, round nuclei can be seen, comprisingone or two nucleoli. The large number of mitochondria in the cytoplasmare slightly domed in shape, showing a few peaks. It is also possible toobserve a number of cytoplasmic granules, which are in fact anaccumulation of glycogen particles organized into a “rosette” asdescribed in the normal liver in vivo (FIG. 5B).

Finally, unexpectedly, the karyotype of the cells of the line issubdiploid. The following karotype formula was deduced: 46 <2n>, XX, +del (7) (q11-q21) inv? (7) (q21 q36), -der (22) t (12; 22) (p11; q11).Determination of the deletion and translocation points was carried outby hybridization in situ. Out of 40 mitoses studied, 65% contain 46chromosomes. 100% of the cells have the following anomalies (FIG. 6):

-   -   a supernumerary and modified chromosome 7 leading to a trisomy        7, and    -   a translocation t (12.22) with a loss of the fragment 12p        leading to a monosomy 12p (FIG. 6).

Other isolated anomalies were detected and are reported in Table Ibelow:

TABLE 1 Cytogenetic Characteristics of the HepaRG Line CHROMOSOMES WITHANOMALIES FREQUENCY (% OF CHROMOSOME ANOMALIES MITOSES) 7?inv(7)(q21q36) Partial trisomy 100 del(q11-q21) supernumerary 12Unbalanced Monosomy 12p 100 translocation 22 t(12; 22)(p11; q11)Monosomy 22p 100 2 supernumerary Trisomy 15 4 supernumerary Trisomy 7.58 i(8)(q10) Monosomy 8p 5 Monosomy 8q

EXAMPLE 2 Functional Ability of the Line

1. Materials and Methods

Extraction of the RNA and Analyses

THE total cell RNA is extracted using the Total SV RNA® kit; (Promega,France), separated on 1.5% agarose gel and analyzed by Northern blot. Acheck on the quantity of RNA transferred to the filters is carried outafter staining with methylene blue.

The hybridization is carried out according to the protocol of Gripon etal. (1993, Virology, 192: 534-540).

Enzyme Activities Associated with the Detoxification Function

The enzyme activities were established by means of the protocolsdeveloped by Guillouzo et al. (1993, Hepatology 18: 406-414).

The following were studied:

-   -   the deethylation of phenacetin to paracetamol,    -   the oxidation of nifedipine to 3,        5-dimethoxy-carbonyl-2,6-dimethyl-4-(2-nitro-phenyl) pyridine,    -   the demethylation of dextromethorphan to dextrorphan tartrate,    -   the hydroxylation of tolbutamide to hydroxytolbutamide,    -   the hydroxylation of 4-mephenyloin to 4-hydroxymephenyloin.

For comparison, enzyme activities were also determined on cultures ofnormal human hepatocytes incubated for 2 to 16 hours in the presence ofthe following substrates:

-   -   2.10⁻⁴ mol/L of phenacetin    -   2.10⁻⁴ mol/L of nifedipine    -   2.10⁻⁴ mol/L of dextromethorphan and mephenyloin    -   1 mmol/L of tolbutamide.

The assays are carried out by high performance liquid chromatography(HPLC). The results are expressed in picomoles of metabolites formed perhour and per μg of DNA. The activity of the glutathion-S-transferase(GST) is estimated using 1-chloro-2, 4-dinitro-benzene (CDNB) (Merck) assubstrate, at pH 6.5 and at ambient temperature.

2. Results

The level of differentiation of the cells was checked by analysis of thequantities of different mRNAs specific to the liver, in particular themRNAs of the proteins of the functions specific to the liver, such asthe proteins of the serum (albumin, transferrin), a hepatic enzymeinvolved in glycolysis (aldolase B) and 3 specific enzymes involved indetoxification (CYP2E1, CYP3A etGSTa). All these RNAs are expressed byadult hepatocytes.

Their expression levels in the proliferation and differentiation phaseswere compared. Moreover, a comparison was established with thecorresponding expression levels in human hepatocytes and the HepG2cells.

FIG. 7 is a Northern blot analysis of the expression of the messengerRNAs in 2 liver biopsies, HepG2 cells and HepaRG cells. The cells weremaintained either under proliferation conditions (prolif) or atconfluence for 1 month. Treatment of the cells with 2% of DMSO for thelast 15 days of culture is indicated as follows: +D.

Little or no specific hepatic mRNAs were detected in the cells duringproliferation. In contrast, these can be detected in the cells atconfluence, maintained for 2 weeks in the presence of a high corticoidconcentration. Those of albumin and aldolase B are strongly expressed,whereas CYP2E1 and CYP3A4 remain weakly expressed. It is also observedthat in the cells having acquired the typical organization intotrabeculae, exposure to DMSO causes an increase in the transcriptscorresponding to the increased expression of the latter two enzymes,making their expression level practically equal to that observed incells originating from biopsies.

The study carried out in parallel on cells of the HepG2 line used as areference reveals that 3 of the 5 specific functions studied are onlyslightly expressed or not expressed in these cells. Moreover, the actionof the DMSO on the HepG2s seems to inhibit the expression of certain ofthese functions, in particular CYP2E1 and CYP3A.

These analyses demonstrate the original character of the HepaRG line.

The activity of these different enzymes corresponding to detoxificationphases I and II was then measured. The other cell lines weakly expressthese enzymes, often late, or also do not respond to specific inducers.

The activity of these different enzymes was measured on confluent HepaRGcells treated with DMSO. This activity was compared to that of primarycultures of human hepatocytes, as well as to that of the HepG2 lines andthe BC2 clone of the HBG line. The results are given in Table II below:

TABLE II Activity of the Enzymes of Phases I And II HUMAN PRIMARYCULTURE HEPATOMA OF HUMAN LINE HEPATOCYTES ENZYME ACTIVITY HepaRG HBGBC2 HEPG2 Min/Max Average n Phenacetin deethylase* 0.33 0.1 0.3 0.1-253.9 47 Tolbutamide hydroxylase 0.51 0.03 <0.2  0.2-2.1 0.9 8S-mephenytoin hydroxylase* 0.45 <0.1 ND 0.1-2  0.7 10 Dextromethorphandemethylase* 0.06 0.02 <0.1 0.1-2  0.5 10 Nifedipine oxydase* 1 1 <0.50.5-30 5.7 34 Paracetamol glucuronyl transferase* 3.7 1.5 <0.3 0.3-164.1 26 Paracetamol sulphoconjugation* 0.7 0.16 0.3 0.1-14 3.6 27Glutathion S-transferase+ 0.04 0.008 ND 0.03-0.5  0.12 27 (substrate:chlorodinitrobenzene) *activity expressed in nanomoles of metabolitesproduced/h/mg of proteins +activity expressed in units/mg of proteins

Examination of this table shows that the HepaRG cells possess the enzymeactivities associated with the detoxification function at levelspractically equivalent to those expressed by normal human hepatocytes,except for dextromethorphan demethylase, the activity of which isslightly less. Activation of the phenacetin deethylase and nifedipineoxydase is greater than that obtained with the other lines. Thisactivation is lower than that observed in normal human hepatocytes,whilst remaining within the same order of magnitude (FIG. 8, theactivities were measured after 72 hours of treatment and are expressedas a ratio of the cells treated to the cells not treated correspondingto the control cells).

EXAMPLE 3 Cooperation Necessary Between the Corticoid and DMSO forComplete Differentiation

Experiments were carried out in order to verify whether both agents werenecessary to induce complete differentiation.

1. Effect on the Differentiation

A first series of experiments was carried out in order to study theeffects of the absence of the corticoid on cells having always beenmaintained in its presence. In FIG. 9A, the HepaRG cells at passage 13,were kept for 2 additional passages either in the continuous presence ofhydrocortisone (growth: HN), or in the absence of hydrocortisone(growth: HO). They were then induced to differentiate (diff.) for 3weeks in the absence of hydrocortisone (HO) or in the presence ofhydrocortisone 5×10⁻⁵ M (HN), and in the presence of differentconcentrations of DMSO.

The withdrawal of the corticoid was carried out at confluence, i.e. atthe time when the cells begin their differentiation.

After culture for 2 weeks, in the absence of DMSO, no specific liverfunction (no transcript), except albumin, is detected. The addition of2% of DMSO is so toxic that its use is impossible. The addition of 1% ofDMSO, less toxic, does not induce any more advanced differentiation.

A second series of experiments made it possible to study the effects dueto the absence of DMSO.

When the cells are exposed only to corticoid, the presence of a fewtranscripts (of albumin, transferrin and aldolase B) is detected at verylow levels. The addition of 1 to 2% of DMSO to the culture medium causesa rapid accumulation of a number of transcripts, in particular thosealready mentioned and those of CYP2E1 and CYP3A.

This demonstrates that the cooperation between the corticoid and theDMSO is indispensable in order to reach maximum differentiation.Finally, the stability of the cultures was established over at least 6weeks.

2. Effect on Proliferation: Continuous Use of Corticoid

The cells are kept in a culture medium without corticoid and theirability to carry out differentiation is tested after 2 (FIG. 9A) and 10passages (FIG. 9B).

In both cases, no differentiation is observed and only the mRNA of thealbumin is detected. The addition of 2% of DMSO proves highly toxic andthe addition of 1% of DMSO causes no perceptible changes.

The addition of corticoid only at confluence, on the other hand, causesan accumulation of the different transcripts specific to the liver. Butthis differentiation is incomplete as the CYP2E1 and CYP3A transcriptsare difficult to detect, even after the addition of 1 or 2% of DMSO, thelatter dosage proving to be highly toxic after 10 weeks withoutcorticoid.

Finally, the cells without corticoid gradually undergo growthmodifications: contact inhibition is delayed to the extent that theconfluence plateau has a cell density twice that noted with cellscontinuously maintained in the presence of corticoid (FIG. 3).

This study clearly shows the importance of the cooperation between theDMSO and the corticoid, both in the proliferation phase (the corticoidmaking it possible to reduce the toxicity of DMSO) and in thedifferentiation phase (where the action of the two agents iscomplementary).

EXAMPLE 4 Abilities of the HepaRG Cells to Evolve Towards the Biliaryand Pancreatic Differentiation Routes

1. Materials and Methods

HepaRG cells were separated, diluted to ⅕ in Williams' E medium to which5 μg/ml of insulin, 100 U/ml of penicillin, 100 μg/ml of streptomycin,510-5M of hydrocortisone hemisuccinate, 2 mM of L-glutamine and 10% FCSwere added, then redistributed into wells with a plastic support. Fourculture conditions were produced:

-   -   HepaRG cells were treated with 3.75 mM sodium butyrate in the        maintenance medium as from the day following subculture        according to the protocol described by Blouin et al. (1995,        Specialization switch in differentiating embryonic rat liver        progenitor cells in response to sodium butyrate, Exp. cell res.,        217: 22-33). The medium is then replenished every 2-3 days,    -   the HepaRG cells were treated 5 days after subculture by 3.75 mM        sodium butyrate or 2% DMSO. The medium was replenished every day        for 5 days,    -   the HepaRG cells were treated at strong confluence by 3.75 mM        sodium butyrate; the medium was replenished every 2-3 days,    -   the HepaRG cells were seeded on a plastic support or monolayer        of rat primitive biliary epithelial cells in MEM alpha medium        complemented with L-glutamine (2 mM), i-inositol (0.2 mM), folic        acid (20 mM), β-mercaptoethanol (10⁻⁴ M), transferrin 200 μg/ml,        12.5% FCS and 12.5% horse serum. In certain situations,        cytokines such as LIF, IL-3, SCF or G-CSF were added to the        medium. This medium was replenished every 2-3 days.        Indirect Immunohistochemistry

The cells after washing with PBS are fixed by a solution of 4%paraformaldehyde buffered by 0.1 M sodium cacodylate at pH 7.4 for 20minutes at 4° C. They are then preserved in a PBS buffer until the timeof the analysis.

The specific sites are saturated for 45 minutes with PBS containing 10%FCS. The samples are then treated with primary antibodies for an hour atordinary temperature in PBS containing 0.05% saponin. After 3 washingsin PBS containing 0.05% saponin, the samples are incubated with thesecond antibody coupled to peroxydase. Two washings are then carriedout, then the peroxydase activity is revealed by incubation with 0.4mg/ml of 3,3′-diaminobenzidine in a solution of 0.05M Tris at pH 7.6,0.01% H₂O₂ at 110 volumes.

2. Results

a/ Differentiation Towards the Biliary Route (FIG. 12)

Morphological Modification

Before any treatment and at confluence, the HepaRG cells constitute apopulation which is not very homogenous, where polygonal cells andelongate cells are found (FIG. 12).

Under all the conditions of treatment at confluence of the HepaRG cellsby the sodium butyrate, the cells have the particular morphology ofbiliary cells in culture (FIG. 12). The cells spread out and increase insize, they have a clear cytoplasm with an ovoid nucleus containingseveral nucleoli. The contour of the cells is generally poorly defined.Sometimes, lipid droplets are observed. When the cells are treated atvery strong confluence, certain of the cells die. These cells probablycorrespond to the cells already committed towards the hepatocyte route.

The effect of the butyrate was also tested on HepaRG cells at a lowdensity and in active proliferation phase. A characteristic of thesecells is their ability to proliferate in the presence of a high dose ofsodium butyrate (FIG. 13A).

During the proliferation phase, the HepaRG cells form a homogenouspopulation of epitheloid-type cells, at confluence they have theappearance of biliary epithelial cells.

Phenotypical Changes

The morphological modifications observed in the presence of sodiumbutyrate are correlated to phenotypical changes (Blouin et al. 1995). Weused four markers of differentiation towards the biliary route,γ-glutamyl transferase, the α6 chain of integrins and the cytokeratins 7and 19 which normally disappear when the cells are directed towards thehepatocyte route. For differentiation towards the hepatocyte route, welooked for an increase in the expression of albumin and a reduction inthe expression of the al chain of the integrins.

In the first instance we characterized the non-differentiatedproliferating cells. The cells of the HepaRG line have the followingphenotype: expression of the c-kit, cytokeratins 7 and 19, α1 and α6chains of the integrins, γ-glutamyl transferase and albumin suggestingthat these cells are oval cells and/or stem cells (see diagram FIG. 1).

When the cells are cultured in the presence of sodium butyrate, theyexpress γ-glutamyl transferase more strongly, preserve the expression ofthe α6 chain of the integrins, and the cytokeratins 7 and 19 attestingto their commitment towards the biliary route.

When they are cultured in the presence of DMSO, which induces hepatocytedifferentiation, we observe a disappearance of the expression of thecytokeratins 7 and 19, α6 chain of the integrins. They express the α1chain of the integrins at a lesser level and albumin very strongly,confirming their hepatocyte differentiation. The cell populations beingheterogeneous in the cultures, these highly characteristic phenotypesare found only in the plaques differentiating towards one or other ofthe routes.

The results obtained after in situ marking of the cells treated or nottreated by sodium butyrate are summarized in Table III below.

TABLE III Biliary/Hepatocyte Route Differentiation HepaRG HepaRG cellscells HepaRG 2% DMSO Sodium cells Differentiation butyrate beforehepatocyte Differentiation treatment route biliary route γ-glutamyl +−/+ ++ transferase α6 chain + − + Cytokeratin 7 + − + Cytokeratin 19 ++− ++ Albumin −/+ ++ −/+ α1 chain ++ + −/+ −/+: weakly positive; +positive; ++: highly positiveb/ Differentiation Towards the Pancreatic Route (FIG. 13B)

As illustrated by the diagram in FIG. 1, the “oval” stem cells of theliver are pluripotent. Apart from being able to differentiate intohepatic cells, which is demonstrated above, they have the ability todifferentiate into other cell types, including the pancreatic cell ofthe acini. We researched whether this differentiation potential was oneof the characteristics of the HepaRG line.

When the cells are seeded and cultured in MEM alpha culture medium, thecells adhere, then retract in order to former spheroid structuressuggesting the formation of complex canalicular networks,morphologically similar to the three-dimensional cultures of pancreaticepithelial cells (Keer-Conte et al., 1996, Ductal cyst formation incollagen-embedded adult human islet preparations: a means to thereproduction of nesidioblastosis in vitro, Diabetes, 45:1108-1114).

EXAMPLE 5 Infection of the Line by the HBV

1. Materials and Methods

Infection

The infectious source is preferentially constituted by the supernatantof HepG2 cells of the clone 2.2.15, concentrated 100 times. In fact,this infectious source has the double advantage of being inexhaustibleand of constant quality.

The cells used for the infection are cells having been culturedaccording to the conditions defined in Example 1 (permanent presence of5.10⁻⁵M of hydrocortisone hemisuccinate) and having been maintained fora week at confluence, then for 2 weeks in the same medium with 2% ofDMSO added.

These cells are incubated with the infectious source, diluted 10 times,in the culture medium with 4% of PEG 8000 (Sigma) added, for 20 hours at37° C. The second part of this HBV infection protocol was established byGripon et al., 1993, Virology, 192: 534-540.

The control cultures were incubated with 4% of PEG and 25% of FCSdiluted in a solution of phosphate buffer saline (PBS).

At the end of the incubation, the cells are washed three times withculture medium and maintained in the presence of 2% of DMSO and 5.10⁻⁵ Mof hydrocortisone hemisuccinate until they are used.

Neutralization tests were carried out before infection by incubating thevirus with hepatitis B surface monoclonal antibodies (S39-10) for 1 hourat ambient temperature.

Detection of the Hepatitis B Virus Surface Antigen (HbsAg)

The HBs antigen was detected in the medium using the ELISA kit (MonolisaAgHBs plus®) from Biorad Laboratories. The results are expressed inpg/ml of supernatant.

Extraction of Viral DNA and Analyses

Viral DNA replication intermediates were isolated in all the celllysates. The cells are recovered after separation with trypsin, thenlysed at 37° C. with a lysis buffer (10 mM of Tris-HCl pH 7.4, 0.5% ofSDS, 10 ml of EDTA pH 7.4, 10 ml of NaCl) with proteinase K (200 μg/ml)added. The cell DNA is precipitated over 12 hours, at 4° C. with 1MNaCl. The supernatant containing the viral DNA is then extracted. Thecomplete viral particles are isolated from the cell supernatant byimmunoprecipitation with an anti-HBs polyclonal antibody (Dako, France).The nucleic acids are finally extracted after 12 hours of lysis at 37°C., in a lysis buffer with tRNA (40 μg/ml) and proteinase K (200 μg/ml)added.

In all the above protocol, the DNA is extracted using phenol-chloroformand precipitated by isopropanol.

The nucleic acids are analyzed by Southern blot on a 1.5% agarose gel.The molecular weight markers are restriction fragments of the DNA of thehepatitis B virus (3182 and 1504 bp). The hybridization is carried outaccording to the protocol of Gripon et al. above.

2. Results

Evidence of the Infection

The intracellular viral DNA is analyzed over the 10 days following theinfection.

FIG. 10A shows the effects of PEG on the infectability of the cells. Itis a Southern blot analysis of the kinetics of appearance of theintracellular viral DNA after infection of the HepaRG cells in thepresence of 5% of PEG (+PEG), or in the absence of PEG (−PEG). Theposition of the relaxed circular (RC) DNA and covalently closed circular(CCC) DNA forms are indicated to the right of the figure. The migrationposition of the molecular weight markers (fragments of the genome of thehepatitis B virus) is indicated to the left of the figure.

In the absence of PEG, the DNA profile observed is identical to thatrevealed for the viral particles present in the inoculum.

However, this signal gradually disappears until becoming practicallyundetectable 10 days after the infection. Moreover, no intermediate formof viral replication is detected under these conditions. On the otherhand, the latter are clearly present when the cells are infected in thepresence of PEG. A strong signal is observed immediately after theinfection, with a profile similar to that observed in the absence ofPEG. This proves that the penetration of the viral particles into thecells is effective. On the second day, the signal is sharply reduced dueto the elimination of numerous viral particles, but a fine bandpositioned towards 2 kb can be observed. This band corresponds to CCCDNA (covalently closed circular DNA). The signals detected atintermediate positions correspond to nascent viral DNA, graduallyincreasing until the 10th day.

In parallel, the kinetics of viral RNA accumulation is established. FIG.10B is a Northern blot analysis of this kinetics after infection of theHepaRG cells, the HepG2 cells are used as a negative control of theviral infection. The size of the RNAs is indicated to the left of thefigure.

The signals at 2.4 and 3.5 kb, corresponding to the main species of RNAspecific to the HBV, appear after two days of infection and accumulateonly in the infected cells.

In order to verify the specificity of the HepaRG cells to be infected,infection of two other hepatoma lines, HepG2 and BC2, was carried outunder identical conditions. These two lines were selected because theyare capable of differentiating. They were cultured under conditionsallowing them to reach a maximum differentiation stage, as well as underthe same conditions as those used for the HepaRG cells. Afterapproximately 12 hours of incubation with the viral particles and 5% ofPEG, the intracellular viral DNA and the viral RNA produced areanalyzed. Immediately after infection, a large quantity of viral DNA isdetected in all the lines, and above all in HepaRG and BC2. To theextent that the trypsin/EDTA treatment eliminates the viral particlesadsorbed at the surface of the cells, the DNA observed must correspondto viral particles having penetrated to the interior of the cells. Thesignal decreases considerably in the two days following the infection.On the other hand, 18 days after the infection, only the HepaRG line hasforms corresponding to viral replication intermediates i.e. the ccc DNAand the viral RNA forms. This shows that only the HepaRG line is capableof being infected and initiating the replication of the virus.

The production of viral particles was sought in the supernatant of theinfected HepaRG cells. The HBs antigen was assayed by radioimmunology.FIG. 11A represents a kinetics of AgHBs secretion in the supernatant ofthe cells infected. The supernatants were collected for 15 days afterthe infection of the HepaRG cells (▪), HBG BC2 (

) and HepG2 (□).

A high HBs antigen concentration was detected in the supernatant. Theantigen concentration increased during the 9 days following theinfection, and was then maintained at a high level. On the other hand,in the supernatant of the HepG2 et BC2 cells, the antigen was onlydetected in the two days following the infection, which shows that theviral particles were adsorbed by the cells, then gradually released intothe medium.

Moreover, the complete viral particles present in the medium wereestimated by immunoprecipitation with an anti-HBs antibody, followed bya Southern blot analysis of the DNA present in the precipitate. FIG. 11Bshows this Southern blot analysis of the appearance kinetics of theextracellular viral DNA in the supernatant of the HepaRG cells infectedby the HBV. After immunoprecipitation of the complete viral particleswith an anti-HBs antibody, the viral DNA is extracted, then analyzed.The migration position of the molecular weight markers (fragments of thegenome of the hepatitis B virus) is indicated to the right of thefigure.

A strong signal is observed two days after the infection. On the 4thday, it has decreased considerably but it gradually increases againuntil the 8th day and is maintained throughout the culture period. Thiskinetics analysis is suitable for the method of replication of thevirus: after a period of release of the viral particles havingpenetrated the cell (for 2 to 4 days), the virus begins to replicateactively. The profile observed differs slightly from that obtained onthe 2nd day.

In Vitro Neutralization of the Infection

In order to determiner whether a specific anti-S monoclonal antibody iscapable of neutralizing the infection of the cells, virions areincubated with variable concentrations of antibodies, then brought intocontact with the cells. The residual infection is estimated as afunction of the long-term secretion of HBs antigen.

FIG. 11C shows this viral neutralization test in vitro. The viralparticles are incubated with seriated dilutions of a monoclonal antibodydirected against the Hbs antigen (antibody S 39-10) (

) or of a non-relevant antibody (▪) and their infectivity evaluated onthe HepaRG cells. The level of infection of these cells is estimated bymeasuring the secretion of HBs antigen in the supernatant of the cellsinfected, 10 days after the infection.

In this figure, it can be seen that concentrations of 10 and 1 μg/mLblock the viral infection, whilst lower concentrations only inducepartial inhibition. A 50% inhibition corresponds to a concentration of0.03 μg/mL.

Influence of the Degree of Differentiation on the Infection

The ability of the HepaRG cells to be infected was studied as a functionof their degree of differentiation. Using the methods described inExamples 1 and 2, the cell differentiation programme was modified.

A first series of infection was carried out on cells continuallymaintained in the presence of corticoid only, then in the presence ofcorticoid and different concentrations of DMSO, in order to inducedifferent stages of differentiation.

The analyses carried out 2 weeks later on the viral RNA show acorrelation between the accumulation of the two main viral transcriptsand the level of differentiation.

FIG. 10C shows the effects of hepatocellular differentiation oninfection by HBV. It is a Northern blot analysis of the intracellularviral RNAs after infection of the HepaRG cells in the presence ofincreasing concentrations of DMSO.

This correlation was confirmed by a series of experiments carried out oncells infected at confluence (before their differentiation) then placedunder optimum conditions in order to induce their differentiation. Theresults are given in Table IV below:

TABLE IV Effect of Hydrocortisone Hemisuccinate and Cell Proliferationon the Infectability of HepaRG Cells Culture HbsAg secretion (pg/ml)#conditions HN Differentiation HO HN with 2% conditions HO after HO afterHN after of DMSO after Infection 17th day 17th day 17th day 17th day D3(growth) <20 <20 33 41 D5 (growth) <20 <20 37 84 D10 (confluence) <20<20 93 171 #HbsAg secretion measured in the supernatant of the cells 47days after seeding

Examination of this table shows that these quantities of HBs antigenmeasured are low when the cells are infected before treatment by DMSO,which means that an infection carried out on slightly differentiatedcells is not very effective compared with that carried out on stronglydifferentiated cells. Similarly, an infection carried out on cellsmaintained in a medium without corticoid (for 10 passages), thencultured for two weeks in the presence or absence of DMSO, also provesto be ineffective, in particular, for cells not having benefited fromthe action of the DMSO.

Finally, the effectiveness of an infection carried out onundifferentiated cells multiplying actively was compared with that oncells with arrested growth and well-differentiated. The results aregiven in Table V below:

TABLE V Effect of Cell Differentiation on the Infectability of HepaRGcells HbsAg secretion (pg/ml)# Differentiation Infection on dayInfection on day medium 15 day after 28 after (after day 17)differentiation differentiation DMSO 0% <20 31 DMSO 1% 70 214 DMSO 2%79 >2000 #HbsAg secretion measured in the supernatant of the cells 47days after seeding

The results show that an infection carried out on cells in theproliferation phase is ineffective, even in the presence of corticoidand of DMSO.

EXAMPLE 6 Use of the HepaRG Line for the Evaluation of the AntiviralActivity of Chemical or Biological Molecules

1. Materials and Methods

The cells are maintained and infected according to the protocols ofExamples 1 and 5.

The antiviral treatment by 3TC is administered from the 8th day to the15th day following the infection of the HepaRG cells, at finalconcentrations of 0.1 and 10 μM in the culture medium. A completereplenishment of the medium is carried out every two days. Thesupernatants are stored at −20° C. and the cell pellets at −80° C.

The viral antigen (HBs) was detected in the culture supernatant usingthe commercial ELISA test ETI-MAK-3® (SORIN).

The viral DNA present in the culture supernatant could be detected andquantified by the Amplicor-Monitor test (ROCHE). The results areexpressed as number of viral particles/ml. For the analysis ofintracellular viral DNA, the cells were separated with trypsin andstored at −80° C. The total DNA was isolated according to the protocoldescribed previously (Zoulim et al., 1998, Drug therapy for chronichepatitis B virus replication. J. Hepatol. 29:151-168).

2. Results

The HBs antigen is detected in the culture supernatant after 8 days oftreatment by 3TC, the value remaining constant whatever the dose andequal to the non-treated control.

The Amplicor-Monitor® test demonstrates a dose-dependent reduction inthe quantity of viral DNA present in the HepaRG cells infected by theHBV after 8 days of treatment by 3TC, with respect to the control cellsinfected by the HBV and not treated, as shown by the results of Table VIbelow.

TABLE VI Antiviral Activity of 3TC 3TC Concentration μM Inhibition 10100 1 98 0.1 69

EXAMPLE 7 Use of the HepaRG Line for the Evaluation of the AntisepticPower of Products Capable of Inactivating the HBV, for Example JavelWater

1. Materials and Methods

2.

1.1. The HepaRG cells are maintained according to the protocols ofExample 1.

1.2. The infection is carried out in parallel:

-   a) either with a native inoculum obtained as previously described    from the HepG2 2.2.15 cells, the infectivity of which is documented    according to the operating protocol of Example 5,-   b) or with the same inoculum brought into contact with the    inactivating solution to be tested, here Javel water.

The antiseptic power will depend on the concentration and period ofcontact of the product with the inoculum. We tested the Javel water in 2concentrations: 12° et 24° and two exposure periods: 15 s and 30 s asdetailed in the following paragraph.

Exposure of the HBV of the inoculum to Javel water.

-   -   series 1: Javel 12° for 15 seconds    -   ″ 2: Javel 12° for 30 seconds    -   ″ 3: Javel 240 for 15 seconds    -   ″ 4: Javel 240 for 30 seconds    -   ″ 5: distilled H₂O used for diluting the virus: control virus

200 μl of pure virus stock then 200 μl of Javel water are poured into 4ml tubes. The reaction is stopped by adding 1.6 ml of PBS (dilution1/10).

Elimination of Excess Javel.

The above viral preparation is decanted into special tubes (2 tubes perseries) and ultracentrifuged at 4° C. in order that the HBV isprecipitated in the pellet.

The supernatant containing the Javel water is gently drawn off.

The small residual volume of the 2 tubes for each dilution is mixed andthe virus pellets recovered by homogenization.

The more or less inactivated viral suspension of each series isinoculated into HepaRG cell cultures according to operating conditionsidentical to Example 5.

1.3. In order to reveal the infection, both the proteins and the viralDNA will be measured.

The viral Ag (Ag Hbs) is sought by a commercial ELISA test (for example.ETI-MAK-3® from Sorin or also Monolisa Ag HBsplus® from Biorad) asdetailed in Examples 5 and 6.

The viral DNA is sought using PCR in the culture supernatant and can bealso be quantified by the Amplicor-Monitor® test (ROCHE). The resultsare expressed in viral genome/ml equivalents. In order to analyze theintracellular viral DNA, the cells are separated with trypsin and storedat −80° C. The total DNA is isolated and the search for DNA specific toHBV is carried out according to the protocol described previously(Zoulim, 1998).

2. Results

2.1. The control inoculum without Javel water makes it possible toobtain production of Ag Hbs and viral DNA in the supernatant as well asthe characteristic profiles of the DNA of HBV in the cells, as detailedin Example 5. This infection was obtained with the pure virus anddiluted to 1/10.

2.2. With the inoculum exposed to different concentrations of Javelwater as described above, it appears that, after contact for 15 or 30seconds with 12° Javel water, no infection takes place. The same appliesto the 24° Javel water (15, 305), for a 1/10 dilution of the virus.

3. Conclusions

The test on HepaRG cells therefore makes it possible for the first timeto offer an in vitro infection cell model of the HBV capable ofvalidating physical (heating, irradiation) or chemical procedures(detergent/antiseptic solutions or gases: peroxides, ozone etc.) capableof inactivating this major contaminant virus involved in nosocomialinfections via medico-surgical devices and instruments.

In this example, a single dilution was tested. Experiments in progresswill specify the sensitivity of the model and the number of logs ofinfectious doses (expressed as a logarithm to the base 10) that it ispossible of evaluate including the capacities of concentrations of thevirus by ultracentrifuging/filtration/precipitation.

EXAMPLE 8 Construction of a “cDNA Chip” from the HepaRG Line

This construction comprises the following stages

-   -   Preparation of two total RNA pools, then purification of poly A        RNA on the one hand, from cells originating from the        differentiated HepaRG line after culture in a medium containing        10⁻⁵ M of hydrocortisone hemissuccinate then 2% of DMSO, and on        the other hand of non-differentiated cells taken 5 days after        subculture.    -   Preparation of complementary DNAs, by reverse transcriptase.    -   Carrying out suppressive subtractive hybridization using a        commercial kit.    -   Cloning of the cDNAs present in the subtractive library.    -   Test of the representativeness of the library by sequencing of a        limited number of cDNAs.    -   PCR amplification of the cDNA products of interest using the        universal primers present in the vector.    -   “Spotting” of the PCR products according to one of the        “microarray”-type processes.

Thus the expression of 1000 to 2000 genes representative of functionalstates of the expression of hepatic cells can be studied in varioussituations of interest.

EXEMPLE 9 Inoculation and Culture of the Hepatic Forms of Plasmodiumfalciparum in the HepaRG Line

A few years ago, it was demonstrated that normal human hepatocytes inprimary culture represented a system favourable to the survival ofPlasmodium falciparum and that the latter could support the completehepatic cycle of the parasite. In contrast, replication models in thehepatoma cells have to date been lacking.

The novel HepaRG hepatoma line may represent an important advance, dueto observation in a test involving infestation of these cells by theparasite, of a complete cycle with formation of schizonts.

Materials and Methods

P-falciparum sporozoites are prepared from Anopheles stephensimosquitoes infected by ingestion of blood cells, themselves infected bythe erythroid forms of the parasite, the gametocytes.

Approximately two weeks after the infectious meal, the infected salivaryglands are dissected under aseptic conditions and collected in culturemedium.

These suspensions, i.e. 10 pairs of salivary glands per 100 ml ofmedium, are added to the cultures of HepaRG cells.

The cells used for the infestation were cultured according to theconditions defined in Example 1 (permanent presence of 5.10⁻⁵ M ofhydrocortisone hemisuccinate) and maintained for a week at confluence,then for 2 weeks in the same medium with 2% of DMSO added.

At the time of the infestation, they are confluent and completelydifferentiated by the corticoid/DMSO treatment described in Examples 1to 4.

The exposure or inoculation period is approximately 3-4 hours and iscarried out in the same medium deficient in DMSO. Then, medium is addedto the cultures and replenished every 2-3 days.

Detection of the Parasites

The preparations are washed in PBS then fixed and stained byMay-Grunwald Giemsa staining. The intra-cellular parasites have acytoplasmic localization. The characteristic formation of schizontsappears clearly. These schizonts firstly have a small number of nuclei,then they grow larger, with a considerably increased number of nuclei,attesting to the realization of a complete replication process.

Optimization of the Infestation Protocol

Plasmodium falciparum, a very fragile parasite, has appeared highlysensitive to DMSO. The results, showing a greater effectiveness, wereobtained by removing the DMSO from the culture medium during theinoculation period.

EXAMPLE 10 Injection of hepaRG Cells into a Mouse

It is possible to inject hepaRG cells into an immunodeficient laboratoryanimal such as a nude mouse in order to obtain an in vivo study model.

This injection into the mouse allows the hepaRG cells to find anenvironment favourable to their proliferation and their differentiation,leading to the formation of a large mass of differentiated cells. Giventhe pluripotent character of the hepaRG cells, these cells will undergodifferent differentiations depending on the cell implantation site(hepatic, pancreatic cells etc.). This injection thus makes it possibleto reconstruct an organ of hepatic or pancreatic type in an animal,having an alteration of said organ (see article of M. Dandry et al.,2001, 33: 981-988, Hepatology, Repopulation of mouse liver with humanhepatocytes and in vivo infection with hepatitis B virus).

This model not only allows the study of the functions of the humanhepatocyte in a whole animal organism but also the study of theinfection of human hepatic cells by hepatotropic viruses and/orparasites in an animal model.

EXAMPLE 11 Infection of the Line by the HCV

1. Materials and Methods

Infection

The infectious sources originate from serums of patients infected by theHCV. At present this is the only source of virus available, knowing thatto date it has not been possible to develop any virus production model.Five serums were tested. The viruses from two serums (Nos. 2 and 4) havea genotype lb and that of serum No. 3 is a virus of genotype 3. Thegenotyping was not carried out on the two serums used, it concernsserums No. 1 and No. 5.

The cells used for the infection were cultured according to theconditions defined in Example 1 (permanent presence of 5.10⁻⁵ M ofhydrocortisone hemisuccinate) and maintained for a week at confluence,then for 2 weeks in the same medium with 2% of DMSO added.

These cells are incubated for 48 hours at 37° C. with the infectioussource, diluted 10 times, in the culture medium devoid of FCS andoptionally with PEG 8000 (Sigma) added.

For controls, cultures were incubated under identical conditions but theserum used this time originates from a patient not infected by the HCV.

At the end of the incubation, the cells are washed three times withculture medium and maintained in the presence of 2% of DMSO and 5.10⁻⁵ Mof hydrocortisone hemisuccinate until they are used.

Extraction of the Intra and Extracellular Viral RNAs and Analyses

The replicative forms of the intracellular viral RNAs were isolated inall the cell lysates. The cells are recovered after separation withtrypsin, then the total RNA is extracted using the High Pure RNAIsolation kit (Roche). The quantity of total RNA extracted is estimatedby optical density in order to standardize the different samples. Thequality and homogeneity of the RNAs are examined by visualizing theribosomic RNAs (rRNA: 28S and 18S) on a 1% agarose gel stained withethidium bromide.

The RNAs of the viral particles are extracted from the culturesupernatant using the High Pure viral RNA kit (Roche).

By retrotranscription (RT), the complementary DNA (cDNA) of the viralRNAs of positive polarity is synthesized from a specific primehybridizing at 5′ of the region which codes for the viral capsidprotein. This oligonucleotide (5′-TTTGAGGTTTAGGATTYGTGCTCAT-3′),designated as SEQ ID NO:1, derived from that described by Martell etal., 1999, Journal of Clinical Microbiology, 37: 327-332 , designatedC-342. The cDNA is then amplified by the “Polymerase Chain Reaction”(PCR) technique using two primers hybridizing in the 5′ non-translatedregion of the viral genome. The primers used are as follows: the senseoligonucleotide (5′-TGAGTGTCGTRCAGCCTCC-3′), designated as SEQ ID NO:2,and the antisense oligonucleotide (5′-ACCACAAGGCCTTTCGCRACCCAC-3′),designated as SEQ ID NO:3, which corresponds to that conceived byMercier et al., 1999, Journal of Virological Methods, 77 1-9, designatedNCR-3. The amplification product (amplicon of 190 pb) is visualized on a2% agarose gel stained with ethidium bromide. A marker of molecularweight of DNA (M) is also deposited in order to verify the identity ofthe amplicon by its size. The effectiveness of the different stages isalways controlled by detecting the viral RNA present in a standard (T+),which corresponds to a diluted serum, originating from an infectedpatient. The absence of any contamination is also systematicallyverified in the retrotranscription (RT) and amplification (POR) stagesby substituting water (T-) for the samples.

2. Results

Evidence of the Infection

FIG. 14 shows the analysis of the infectability of the HepaRG cells byviruses originating from the five serums tested. This involves lookingfor the presence of the viral RNA both in the extracellular andintracellular compartments by RT-PCR after infection of the cells in thepresence of PEG 8000. The viral RNA is sought on the 12th day followingthe infection. The position of the amplicon is indicated to the right ofthe figure. The rRNAs are shown after homogenization of the samples inthe lower part of the figure where the position of the 28S and 18S RNAsis indicated on the right.

During the use of a inoculum without any virus (HCV-serum), no viral RNAis detected either in the culture supernatants or in the cells. Despitethe origin of the line which was selected from a liver tumor taken froma female patient suffering from viral hepatitis C, no residual viralreplication is detectable in the HepaRGs.

The semi-quantitative technique of detection by RT-PCR makes it possibleto reveal the presence of signals not only in the intracellularcompartment but also in the culture supernatants whatever the serum used(serums No. 1 to No. 5). These results explain the establishment of anHCV replication in the HepaRG cells which leads to an effectivesecretion of virions by the cells.

In order to confirm that a viral replication is established in theHepaRG cells after their infection, the sensitivity of the HCVreplication to an antiviral, interferon α, was studied. The cells wereinoculated over 48 hours in the presence of PEG 8000 by thevirus-containing serum No. 4. A serum without any HCV is used as anegative control of the viral infection. Cytokine (Introna®,Schering-Plough, France) is added to the culture medium from the end ofthe 6th day to the 12th day following the infection of the cells, atfinal concentrations of 5, 50 or 500 U.I./ml. Complete replenishment ofthe medium is carried out every two days. The supernatants are stored at−80° C. for kinetics analysis.

FIG. 15 shows the incidence of the presence of the interferon α on the12th day following the exposure of the cells to the infectious source,with respect to the quantity of viral RNA detectable in the cells and inthe supernatants. The position of the amplicon is indicated to the rightof the figure. The rRNAs are shown after homogenization of the samplesin the lower part of the figure where the position of the 28S and 18SRNAs is indicated on the right. Starting from the lower dose (5U.I./ml), a drop of more than 60% of the intracellular signal isdetected after the six days of treatment and complete disappearance ofthe signal is obtained in the extracellular medium.

Kinetics of HCV Replication and Its Inhibition by Interferon α

A kinetics of HCV replication and of its inhibition by interferon α wasestablished and is shown in FIG. 16. The presence of the viral RNA inthe supernatants was sought in parallel in a non-treated referenceculture (ν) and in a culture where the cytokine is present in the mediumat 5 U.I./ml a concentration the effectiveness of which was previouslyverified (FIG. 15), from the end of the 6th day following the infection,until the 12th day after exposure to the infectious source (ο).

Analysis of the sample taken at the end of the 2nd day following theinfection (ν, Day 2) reveals a strong signal. This phenomenon mustcorrespond to a release from the inoculum of the viruses which wereinitially adsorbed onto, or internalized in, the cells. Then, theconstant signal detected from the 4th day post-infection (υ, Day 4 toDay 12) translates an active replication of the virus which leads to aconstant secretion of virions over the period studied. The first part ofthe kinetics carried out on the cultures incubated in the presence ofinterferon α is identical to that carried out in the absence of theantiviral (compare υ and ο from Day 2 to Day 8). This makes it possibleto affirm that a replication was well established in the HepaRG cellsbefore the treatment. In contrast, after four days of incubation withcytokine, no extracellular viral RNA is detected, until the end of thetreatment (ο, Day 10 to Day 12). The inhibitory action of interferon αobserved in vivo is therefore well reproduced in the HepaRG cellsinfected by the HCV, after a minimum treatment period of 2 days.

EXAMPLE 12 Infection of the Line by Parasites of the Genus Leishmania

The leishmaniases are diseases subsequent to infection by a parasite ofthe genus leishmania. The clinical presentation of these infectionsranges from localized cutaneous infection, to a disseminated infectionduring which the main organs infected are the lymph glands, bone marrow,spleen and liver. The leishmania are transmitted in flagellatepromastigote form by a hematophagous diptrous insect, the phlebotominesand fly, and rapidly become intracellular in their host. The cellscurrently described as permissive to the leishmanias are the cells ofthe mononuclear phagocyte system. However, a hepatocyte infection testrecently demonstrated the permissiveness of this cell type, opening upresearch perspectives in order to better understand the physiopathologyof the infection and to evaluate new therapeutic targets.

1. Materials and Methods

Infection

The Leishmania major, L. donovani, L. infantum, L. tropica, L.braziliensis and L. guyanensis promastigotes used are strains isolatedfrom patients, identified by the reference isoenzymatic method andcryopreserved in liquid nitrogen. Obtaining an inoculum requires atleast 2 successive amplification phases in Novy-McNeal-Nicolle gelosemedium with rabbit blood added, then in Schneider medium with 10% of FCSadded.

The cells used for the infection were cultured according to theconditions defined in Example 1 (permanent presence of 5.10⁻⁵ M ofhydrocortisone hemisuccinate) and maintained for a week at confluence,then for 2 weeks in the same medium with 2% of DMSO added.

These cells are incubated for 18 hours at 37° C. with leishmaniapromastigotes, then washed 3 times with culture medium and maintained inthe presence of 2% of DMSO and 5 M hydrocortisone hemisuccinate.

Detection of Intracellular Parasites

The checking and quantification of the infection are carried out byoptical microscopy. The cells are recovered after trypsinization of thewells and 2 washings in PBS, cytocentrifuqed for 10 minutes at 9000 rpm,then fixed and stained by May-Grunwald Giemsa stain. The intracellularparasites in amastigote form have cytoplasmic localization, measure 3×6micrometres and are easily locatable thanks to their structurecomprising a bluish cytoplasm, and a violet nucleus and kinetoplast. Thenumber of cells infected and the total number of parasites found arecalculated for an average number of 4000 hepatocytes read.

2. Results

Evidence of the Infection

FIG. 17 shows the kinetics of infection observed with an L. donovanistrain and an L. major strain for infection ratios of 25 parasites percell. The cells can therefore be infected with a hepatotropic strain (L.donovani) but also a dermatropic strain (L. major). The number ofamastigotes of L. donovani per 100 cells is comprised between 7 and 45for 4000 cells read (0.175% to 1.125%), corresponding to a percentage ofinfected cells comprised between 0.1 to 0.55%. The number of amastigotesof L. major is comprised between and 6 and 38 for 4000 cells read (0.15and 0.725%), corresponding to a percentage of cells infected comprisedbetween 0.1 to 0, 375%.

Optimization of the Infection Protocol

A first experiment studied the “culture duration” effect on the totalnumber of parasites and the number of cells infected. The results ofthis work carried out with cells infected by L. major (infectionratio=25 parasites per cell) are shown in FIG. 18.

A second experiment aimed at studying the “inoculum” effect on the levelof infection of the cells. L. major was successively inoculated in thecells with ratios of 6, 12, 25, 50, 100 and 200 parasites per cell. Theresults of the cultures on Day 7 are shown in FIG. 19 and show that thenumber of parasites per 100 cells varied from 2 to 218 for 4000 cellsread (0.05% to 5.45%), corresponding to a percentage of cells infectedranging from 0.05% to 1.6%.

In total, it appears that the ideal infection ratio by L. major is 100parasites per cell, and the parasite infection quantification is optimumbetween Day 4 and Day 7.

1. A human hepatoma cell line deposited on 5th Apr. 2001 at theCollection Nationale de Cultures de Microorganismes, Institut pasteur,under No. 1-2652.
 2. A method for infecting a human hepatoma cell linewith a hepatotropic parasite and/or a virus, the method comprising: (1)obtaining the cells of human hepatoma cell line selected for having ahepatocyte-type morphology characteristic of resident liver cells of ahuman; (2) maintaining the selected human hepatoma cell line of (1) in aculture medium comprising at least one cortico-steroid at a non-toxicconcentration, and DMSO in a quantity sufficient to inducedifferentiation; and (3) exposing an infectious source comprising ahepatotropic parasite and/or a virus to the human hepatoma cell line of(2) maintained in the culture medium comprising at least onecortico-steroid at a non-toxic concentration to permit infection of thehuman hepatoma cell line by the parasite and/or the virus, wherein thehuman hepatoma cell line comprises a set of cells derived from a biopsyobtained from a cancerous human liver, the set comprises cells thatexpress a receptor having an affinity for binding a Hepatitis C Virus(HCV) in a native form, and/ or cells that express a receptor having anaffinity for binding a Hepatitis B Virus (HBV) in a native form, so thatthe set of cells are susceptible to infection by HCV and HBV, whereinthe virus is in a native form and is selected from the group consistingof Hepatitis C Virus (HCV) and or a Hepatitis B Virus (HBV), and whereinthe hepatotropic parasite is in a native form and is selected from thegroup consisting of Leishmania genus and Plasmodium falciparum.
 3. Themethod of claim 2,wherein obtaining the cells of human hepatoma cellline, selected for having a hepatocyte-type morphology characteristic ofresident liver cells of a human, comprises: (a) providing a biopsy of asolid tumor removed from a hepatocarcinoma patient; (b) growing apopulation of cells derived from the biopsy in a culture mediumcomprising at least one cortico-steroid at a non-toxic concentrationpresent continuously in the culture medium, during a proliferationphase; (c) adding DMSO, in a quantity sufficient to inducedifferentiation, to the culture medium comprising at least onecortico-steroid exposed to the population of cells so that the cultureis continuously exposed to both, a cortico-steroid and DMSO, during adifferentiation phase; and (d) repeating (b) and (c) as necessary toobtain a subpopulation of cells selected for having a hepatocyte-typemorphology characteristic of resident liver cells of a human.
 4. Amethod for maintaining the stability of a human hepatoma cell line, themethod comprising: continuously exposing the cells of the human hepatomacell line to a medium comprising at least one cortico-steroid at anon-toxic concentration in order to promote the differentiation of thecells of the human hepatoma cell line so that the differentiated cellsdevelop structural and functional properties characteristic of residenthepatocytes and resident biliary cells of a human liver, wherein thehuman hepatoma cell line comprises a set of cells derived from a biopsyobtained from a cancerous human liver, the set comprises cells thatexpress a receptor having an affinity for binding a Hepatitis C Virus(HCV in a native form, and/ or cells that express a receptor having anaffinity for binding a Hepatitis B Virus (HBV) in a native form, so thatthe set of cells are susceptible to infection by HCV and HBV, whereinthe medium further comprises sodium butyrate at a concentrationsufficient to induce the differentiation of the cells of the humanhepatoma cell line into a biliary-type lineage.
 5. The method of claim4, wherein the sodium butyrate is at a concentration of about 2.5 toabout 5 mM.
 6. The method of claim 4, wherein the sodium butyrate at aconcentration of about 3.75 mM.